The long-range goal of this competitive renewal of a previous R01 is to investigate genetic programs that control pain relay sensory neuron phenotypes in the dorsal spinal cord. During the previous funding interval, my colleagues and I have compiled a genome-scale expression map of transcription factors in the mouse nervous system. Subsequent genetic studies demonstrate that Tlx3, a homeobox class transcription factor, is a pivotal regulator of spinal relay sensory neurons, including specification of both glutamate and peptide neurotransmitters. Furthermore, persistent Tlx3 expression in adult animals is confined to superficial laminae, where putative pain relay neurons are located. The research described here builds upon this preliminary work. The goal of our research over the next five years is to illustrate the roles of Tlx3 in regulating spinal relay nociceptor phenotypes and pain behaviors and to gain insights into the molecular and cellular basis underlying pain perception. We have four specific Aims. Aim 1 is to determine the roles of Tlx3 in controlling the development of ascending projection neurons that are critical for pain perception. Aim 2 is to determine how dynamic Tlx3 expression controls lamina organization of the dorsal spinal cord. Aim 3 is determine the roles of Tlx3 in maintaining dorsal horn excitatory neuron phenotypes, thereby determining if Tlx3-mediated core transcription program is a potential target for pain treatment. Aim 4 is to determine the roles of Tlx3-dependent differentiation programs in controlling pain behaviors. Each of these aims is built upon a set of preliminary data that lead to a testable hypothesis. A panel of genetic tools that we have already developed will test the predictions of these hypotheses.